sut-2 loss of function mutants protect against tau-driven shortened lifespan and hyperactive pharyngeal pumping in a C. elegans model of tau toxicity

Expression of human tau in C. elegans neurons causes progressive, age-associated loss of motor coordination, selective neurodegeneration, and shortened lifespan. Loss of function (LOF) mutations in the conserved gene sut-2 protects against progressive motor uncoordination and neurodegeneration in models of tauopathy. To determine whether sut-2 LOF also protects against shortened lifespan of tau transgenic C. elegans , we conducted lifespan assays comparing four different alleles of sut-2 . We found that sut-2 LOF robustly suppresses the shortened lifespan of tau transgenic animals. We also demonstrate that tau transgenic C. elegans exhibit hyperactive pharyngeal pumping, which is restored by sut-2 LOF.

To test whether tau Tg expressing C. elegans exhibit altered pharyngeal pumping, we assayed pharyngeal muscles and neurons electrophysiology from individual animals using a microfluidic chip-based recording device. This device detects, records, and evaluates pharyngeal muscle and neuron action potentials that accompany each pump cycle. We found tau Tg animals had significantly increased frequency and reduced duration of pharyngeal pumping (Fig 1C-F). We then tested whether sut-2(bk3011) and sut-2(bk3012) can modify tau Tg C. elegans pumping defects. We found that sut-2(bk3011) and sut-2(bk3012) partially suppress the increased frequency but do not suppress the decreased duration of pumping in tau Tg animals (Fig 1C-F). sut-2(bk3011) and sut-2(bk3012) do not have altered pumping rates relative to N2 (Fig 1G).
Taken together, these data show that complete elimination of sut-2 via a whole gene deletion ameliorates the toxic consequence of tauopathy in tau transgenic C. elegans. sut-2 and its mammalian homolog MSUT2 may be compelling targets to treat tauopathies including Alzheimer's disease.

Table legend
N = number tested, Mean = average population lifespan in days, p-value (N2) = significance relative to N2 (wild-type control), pvalue (tau Tg) = significance relative to tau Tg animals. N.S. = not significant. Significance was evaluated using survival curve comparisons with Mantel-cox log-rank analysis.

C. elegans lifespan assays
Lifespan assays were modified from those described in (Liachko, Davidowitz et al. 2009). In brief, worms were grown at 25 o C following a short (4-6 hour) egglay to L4 stage on NGM plates seeded with E. coli OP50, and then transferred onto seeded NGM plates with added 5-fluoro-2'-deoxyuridine (FUDR, 0.05 mg/mL) to inhibit growth of progeny. Worms were scored every 1-2 days for movement following tapping of the plate or gentle touching with a platinum wire. Failure to respond to touch was scored as dead. Statistical analysis was performed using GraphPad Prism software.
C. elegans pumping assays C. elegans pumping electrophysiology was evaluated using a ScreenChip System (NemaMetrix/ InVivo Biosystems) as described in (Latimer, Stair et al. 2022).In brief, day 1 adult C. elegans were pre-incubated in M9 buffer containing 10 mM 5HT (Sigma) for 20 minutes, to stimulate pharyngeal pumping (Song and Avery 2012).C. elegans were then individually loaded into the microfluidic recording device and pharyngeal muscle and neuron action potentials were recorded for two minutes using NemAquire software (NemaMetrix/ InVivo Biosystems).Action potential statistics and readouts, including frequency and duration, were computed using NemAnalysis software (NemaMetrix/ InVivo Biosystems). C. elegans pharyngeal pumping rate assay was adapted from (O'Brien 2022). The pump rates were determined by manually counting the number of grinder movements observed in individual animals over 20 seconds. Each animal was recorded 3 times and the average of the 3 recordings was used calculate pumps per minute. Data was managed in Microsoft Excel and statistical analysis was performed using GraphPad Prism Software.